Protein kinase C is one of the largest families of protein kinase enzymes and is composed of a variety of isoforms. Conventional isoforms include α, βI, βII, γ; novel isoforms include δ, ε, η, θ; and atypical isoforms include ξ, and l/λ.
PKC enzymes are primarily cytosolic but translocate to the membrane when activated. Once activated and translocated, PKC is anchored into the membrane by the anchoring protein RACK1. See, e.g., Mochly-Rosen et al. (1991) Proc Natl Aced Sci USA 88, 3997-4000; Nishizuka, Y. (1995) FASEB J 9, 484-496; Sklan et al. (2006) Prog Neurobiol 78, 117-134. RACK1 localizes PKC to its corresponding substrates for phosphorylation, thus making PKC functionally active and physiologically relevant.
Activated PKC participates in a variety of biological pathways. For example, PKC activates ELAV mRNA-stabilizing proteins and cAMP-response-element-binding (“CREB”) proteins. PKC isoforms also play a regulatory role in amyloid precursor protein (“APP”) processing and amyloid accumulation. For example, PKC-α and PKC-ε regulate APP processing by the non-amyloidogenic pathway, suggesting that decreases in these enzymes may lead to increases in A-beta synthesis and accumulation. Thus, PKC activators may be able to reduce levels of soluble A-beta and increase levels of soluble APP-α. PKC activators may also be able to reduce or eliminate amyloid plaques and neurofibrillary tangles.
PKC activators have been associated with prevention and treatment of various diseases and conditions. For example, PKC activators may allow for prevention and treatment of neurodegenerative diseases and conditions, neuroaffective diseases and disorders, cognitive impairments, and diseases and conditions associated with neuronal or synaptic loss. Indeed, PKC activators have been found to induce synapse formation. Moreover, PKC activators have been associated with improvement in, for example, memory and learning, including long-term memory.
In one example, PKC activators have demonstrated neuroprotective activity in animal models of Alzheimer's Disease (“AD”). See Etcheberrigaray et al., Proc. Nat. Acad. Sci. USA, 1992, 89: 7184-7188. AD is a neurodegenerative disorder that is characterized clinically by progressive decline of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately, death.
Pathologically, AD is associated with the accumulation of aggregated β-amyloid (“Aβ”), a 4 kDa peptide produced by the proteolytic cleavage of amyloid precursor protein (“APP”) by β- and γ-secretases. Oligomers of Aβ are considered to be most toxic, while fibrillar Aβ is largely inert. Monomeric Aβ is found in normal patients and has an as-yet undetermined function.
PKC activators can reduce the levels of Aβ and prolong survival of AD transgenic mice. See Etcheberrigaray et al., 1992, Proc. Nat. Acad. Sci. USA, 89: 7184-7188. PKC-ε has been shown to be most effective at suppressing Aβ production. See Zhu et al., Biochem. Biophys. Res. Commun., 2001, 285: 997-1006. Accordingly, isoform-specific PKC activators are highly desirable as potential anti-AD drugs.
The earliest consistent cytopathological change in AD is loss of synapses. See Scheff et al., Neurobiol. Aging, 2006, 27: 1372-1384; and Marcello et al., Eur. J. Pharmacol. 2008, 585: 109-118. In fact, synaptic loss appears to be the only pathological finding in the brain that is closely correlated with the degree of dementia in AD patients. See Terry et al., Ann. Neurol., 1991, 30: 572-580. Evidence suggests that Aβ is involved in synaptic loss.
Other diseases and conditions are associated with synaptic loss and/or Aβ. Persons who have suffered from a brain injury, for example, show increased synthesis and expression of APP and its proteolytic product Aβ. See, e.g., Zohar et al., Neurobiology of Disease, 2011, 41: 329-337; Roberts et al., Lancet, 1991, 1422-1423; Gentleman e al., NeuroReport, 1997, 8: 1519-1522; Iwata et al., J. Neuropathol. Exp. Neurol., 2002, 61: 1056-1068. In animal models, the PKC activator Bryostatin 1 was shown to protect against traumatic brain injury-induced learning and memory deficits. See Zohar et al., Neurobiology of Disease, 2011, 41: 329-337.
Additionally, some forms of stroke are caused by Aβ, such as those associated with cerebral amyloid angiopathy (“CAA”). See U.S. Patent Application Publication No. 2010/0022645 A1. This disorder is a form of angiopathy in which the same Aβ deposits as found in AD accumulate in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessel to failure, increasing the risk of a hemorrhagic stroke. CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down's syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.
Both PKC-α and PKC-ε are important for synaptogenesis—i.e., the formation of synapses. The high abundance of PKC-ε in presynaptic nerve fibers suggests a role in neurite outgrowth, synaptic formation, and neurotransmitter release. See Shirai et al., FEBS, 2008, 29: 1445-1453. Nontoxic drugs activating PKC-α and PKC-ε can promote synaptogenesis under non-pathological conditions and actually prevent synaptic loss under pathological conditions. See Nelson et al., Trends Biochem. Sci., 2009, 34: 136-145; Hongpaisan et al., Proc. Natl. Acad. Sci. USA, 2007, 104: 19571-19576; Sun et al., Proc. Natl. Acad. Sci. USA, 2008, 105: 13620-13625; Sun et al., Proc. Natl. Acad. Sci. USA, 2009, 106: 14676-14680.
For example, PKC activators have demonstrated neuroprotective activity in animal models of stroke. See Sun et al., Eur. J. Pharmacol., 2005, 512: 43-51. Several PKC isoforms play a central role in mediating ischemic and reperfusion damage following stroke. Studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have demonstrated that PKC-ε is involved in induction of ischemic tolerance and prevents damage, while PKC-δ and PKC-γ are implicated in injury. See Takayoshi et al., Stroke, 2007, 38(2): 375-380; and Bright et al., Stroke, 2005; 36: 2781. Postischemic/hypoxic treatment with Bryostatin 1 effectively rescued ischemia-induced deficits in synaptogenesis, neurotrophic activity, and spatial learning and memory. See Sun et al., Proc. Natl. Acad. Sci. USA., 2008, 105(36): 13620-13625
PKC activation has a crucial role in learning and memory enhancement and PKC activators have been shown to increase memory and learning. See Sun et al., Eur. J. Pharmacol. 2005, 512: 43-51; Alkon et al., Proc. Natl. Acad. Sci. USA., 2005, 102: 16432-16437. For example, Bryostatin increased the rate of learning in rodents, rabbits, and invertebrates. See Sun et al., Eur. J. Pharmacol., 2005, 512: 43-51; Wang et al., Behay. Pharmacol., 2008, 19: 245-256; and Kuzirian et al., Biol. Bull., 2006, 210: 201-214. Additionally, Bryostatin-induced synaptogenesis for long-term associative memory was shown to be regulated by PKC activation. Hongpaisan et al., Proc. Natl. Acad. Sci. USA, 2007, 104: 19571-19576.
PKC activation has been associated with a variety of other conditions. For example, PKC activators have demonstrated neuroprotective activity in animal models of depression. See Sun et al., Eur. J. Pharmacol., 2005, 512: 43-51. PKC activators are also associated with prevention and treatment of Parkinson's disease, bipolar disorder, and schizophrenia, mental retardation (and related diseases like autism).
PKC activators can be broad-spectrum activators, acting on multiple isoforms of PKC, or can be selective for certain isoforms. While all types of PKC activators are of interest, selective PKC activators may offer unique advantages because different isoforms perform different, and sometimes opposite, functions. For example, PKC-δ and PKC-θ are often regarded as having a pro-apoptotic function because they are components of the caspase apoptosis pathway. PKC-ε, by contrast, has an opposite role: its activation promotes proliferation and cell survival, and inhibits apoptosis. See Nelson et al., Trends in Biochemical Sciences, 2009, 34(3): 136-145.
A variety of different PKC activators are known. Bryostatin, for example, is a macrolide lactone that competes for the PKC 1,2-diacylglycerol (“DAG”) binding site with very high affinity, producing a brief activation period followed by a prolonged downregulation. Other PKC activators include phorbol esters, naphthalene sulfonamides, and oxidized lipids.
Polyunsaturated fatty acids (“PUFAs”), such as arachidonic acid and 2-hydroxy-9-cis-octadecenoic acid (i.e., minerval), are also known PKC activators. PUFAs are interesting molecules in that they are essential components of the nervous system. They are known to increase membrane fluidity, rapidly oxidize to highly bioactive products, and produce a variety of inflammatory and hormonal effects. In addition, they are of low molecular weight and are able to cross the blood-brain barrier. Further, PUFAs are stable to acid and base, making them potentially effective for oral administration. On the other hand, PUFAs are rapidly degraded and metabolized in the body.
Like PUFAs, certain derivatives of PUFAs have been shown to be PKC activators. For example, certain cyclopropanated PUFAs such as DCPLA (i.e., linoleic acid derivative), AA-CP4 methyl ester (i.e., arachidonic acid derivative), DHA-CP6 methyl ester (i.e., docosahexaenoic acid derivative), and EPA-CP5 methyl ester (i.e., eicosapentaenoic acid derivative) may be able to selectively activate PKC-ε. See Journal of Biological Chemistry, 2009, 284(50): 34514-34521; see also U.S. Patent Application Publication No. 2010/0022645 A1.